Culture Medium

Amoeba Proteose-Yeast Extract-Glucose Broth (PYG) 1. 20 g Proteose peptone (Beckon Dickinson, Franklin Lakes, NJ). 2. Yeast extract (Difco, Detroit, MI). 4. Adjust pH to 6.5 with 1 M HCl and autoclave for 15 min at 121.5 C. 5. After autoclaving, add 50 mL of filter-sterilized 2 M glucose. 6. Add autoclaved and filter-sterilized stock salt solutions of the following 10 mL 0.4 M MgSO4-7H2O, 8.0 mL 0.05 M CaCl2, 10 mL sodium citrate (Na3C6H5O7-2H2O), 10 mL 0.25 M Na2HPO4-7H2O, 10 mL 0.25 M...

Detection and Differentiation of Cryptosporidium Oocysts in Water by Pcrrflp

Lal, and Jianlin Jiang Consumption of contaminated water has been implicated as a major source of Cryptosporidium infection in various outbreak investigations and case control studies. Surveys conducted in various regions of the United States demonstrated the presence of Cryptosporidium oocysts in 67-100 of wastewaters, 24-100 of surface waters, and 17-26.8 of drinking waters (1-4). The identity and human infective potential of these waterborne oocysts are not known,...

Production of Antimicrobial Substances by Lactic Acid Bacteria II

Screening Bacteriocin-Producing Strains With Probiotic Purposes and Characterization of a Lactobacillus Bacteriocin Virginia S. Oca a and Mar a Elena Nader-Mac as 1. Introduction Bacteriocins have been defined as proteinaceous, bactericidal substances synthesized by bacteria, which usually have a narrow spectrum of activity, only inhibiting strains of the same or closely related species (1). The term bacteriocin-like substance is applied to antagonistic substances that are not completely...

Determine Critical Control Points Principle

The Codex guidelines define a critical control point (CCP) as a step at which control can be applied and is essential to prevent or eliminate a food safety hazard or reduce it to an acceptable level (see Notes 1 and 4). 3.4.2. Review of Identified Hazards Prior to determining the CCPs, a review should be conducted to verify whether any of the identified hazards are fully controlled by the application of the Codex General Principles of Food Hygiene, good manufacturing practices (GMPs), or good...

Postelectrophoretic Detection of Esterases

In this method the enzyme extracts (intracellular fraction) are first subjected to a protein separation (electrophoresis in non-denaturing conditions without SDS ), and then lipases and esterases are revealed using a- and p-naphthyl derivates of fatty acids. Released naphthol is coupled with diazotized salts (Fast Red TR salt or Fast Garnet GBC salt) to give a colored product. The PAGE procedure considered here is based on the technique described by Laemmli (5). 3.3.1. Preparation of Separation...

Hemagglutination Ability

Bacterial Cell Suspensions Preparation The suspension of 1 x 109 CFU mL was serially diluted (1 2, 1 4, 1 8, and 1 16) in round-bottomed microplates (see Note 2). 3.4.2. Red Blood Cells Suspension 1. Fresh red blood cells Blood samples obtained by venipuncture with sterile syringes were collected in sterile screw-top tubes by adding 9 vol of blood to 1 vol of sodium citrate solution (3.8 ) (see Notes 4 and 5). 2. Washed red blood cells The blood sample was centrifuged (10 min, 4500g),...

Molecular Genotyping Methods and Computerized Analysis for the Study of Salmonella enterica

Ana Bel n Vivanco, Juan Alvarez, Idoia Laconcha, Nuria L pez-Molina, Aitor Rementeria, and Javier Garaizar Salmonella enterica is widely recognized as a major cause of foodborne diseases in humans and animals and has been isolated from environmental sources in increasing numbers worldwide (1,2). Conventional typing methods such as serotyping and phage typing have been and still are the mainstay in descriptive epidemiology of this microorganism. Nevertheless, limitations on the availability of...

Viruses

Detection of Infectious Rotaviruses by Flow Cytometry Albert Bosch, Rosa M. Pint , Jaume Comas, and Francesc-Xavier Abad Human rotaviruses are considered the main cause of viral gastroenteritis in infants and young children throughout the world (1). Their transmission is through the fecal-oral route, mostly after ingestion of contaminated water and food (2). Since an extremely high number of virus particles are present in the feces during the acute gastroenteritis, methods based on electron...

Info

ALeucocin A is identical to leucocin B (35), piscicocin V1a is identical to piscicolin 126 (36), sakacin A is identical to curvacin A (37), carnobacteriocin BM1 is identical to piscicocin V1b (29), and pediocin PA-1 is identical to pediocin AcH (38), and pediocin SJ-1 (39). Bavaracin A (40) is probably identical to sakacin P. *Residue numbering is according to the sequence of sakacin G. The consensus sequence includes residues conserved by at least 75 . The residues conserved by more than 90...

Notes

Before each growth experiment, one cryovial from the freezer must be removed. The unfreezing procedure should be fast enough to decrease the damage produced by the intra-cellular ice. The microorganisms must always be observed under the optical microscope and also checked for purity. To subcultivate the microorganisms in the broth in which the growth curves are performed, the time and temperature of each subculture must be carefully maintained. To obtain the third preculture, at least three...

And Escherichia coli in the Urinary Tracts of Mice

Clara Silva, Rosario Rey, and Mar a Elena Nader-Mac as The use of probiotic microorganisms has been widely promoted in the last 20 yr (1). They have been used in the gastrointestinal tract as capsules or as fermented milks (2). The characteristics of the strains proposed as probiotics have been published (3) or patented under an elaboration process (4-6). The first step in designing a probiotic product is to isolate and characterize strains with some beneficial properties. The second step is to...

Production of Antimicrobial Substances by Lactic Acid Bacteria I

Mar a Silvina Ju rez Tom s, Mar a Claudia Otero, Virginia Oca a, and Mar a Elena Nader-Mac as Restoration of the balance of different ecological niches has been proposed as a way to control the income of pathogenic microorganisms (1). The genus Lactobacillus has been used in different human and animal tracts as probiotic microorganisms with this objective in mind (2). The characteristics of the strains proposed as probiotics have been published (3) or patented under the process of elaboration...

L J Si

(Fig. 3. continued) profiles of Salmonella Typhimurium isolates digested with XbaI following electrophoresis through 1 (w v) SeaKem Gold agarose in 0.5X TBE for 18 h at 10.5 C. Electrophoresis was performed at 200 V using a Gene Navigator system (Pharmacia) in the interpolation mode pulsing from 1 through 40 s. (B) Following pulsed-field gel electrophoresis the XbaI macrorestricted DNA fragments were transferred to nylon membranes. Individual gene cassettes were then used to probe the...

PrPres and TSE Strains

Strains of TSE agent have been described for CJD (90), scrapie (68,69), and hamster-adapted TME (7). Strains in the TSE are defined by differences in brain pathology, disease incubation time, clinical disease, species tropism, and PrP-res properties (for review, see ref. 19). It is especially challenging to explain TSE strains in terms of PrP-res formation since multiple strains have been described in species with one PrP genotype. It is difficult to imagine how PrP-res with the same amino acid...

RNA Isolation Procedures for Rotavirus see Note

Inactivation of Endogenous Ribonucleases (see Note 2) 1. Diethyl-pyrocarbonate (DEPC). 2. RNase AWAY (Molecular Bioproducts, San Diego, CA). 2.1.2. RNA Extraction SDS Proteinase K Digestion-Phenol Chloroform Extraction 1. 10 mg mL Proteinase K, prepared using DEPC water. 2. 10 (w v) Sodium-dodecyl sulfate (SDS) prepared using DEPC water. 5. 1 1 Phenol chloroform (see Note 3). 6. Sterile 1.5-mL Eppendorf tubes. Fig. 3. Schematic representation of the seminested PCR assay that identifies...

Establish Corrective Actions Principle

Establishing Corrective Actions The Codex Hazard Analysis and Critical Control Point (HACCP) System and Guidelines for Its Application defines corrective action as any action to be taken when the results of monitoring at the CCP indicate a loss of control. Loss of control is considered a deviation from a critical limit for a CCP (47). Process step CCP No. Hazard Deviation HACCP limits procedures procedures records Process step CCP No. Hazard Deviation HACCP limits procedures procedures...

John F T Spencer and Alicia L Ragout de Spencer

Planta Piloto de Procesos Industriales Microbiol gicos (PROIMI)-CONICET San Miguel de Tucum n, Argentina The world abounds with microorganisms that are hazardous to human health in the normal course of life. In Public Health Microbiology Methods and Protocols, expert investigators describe in step-by-step detail the laboratory techniques they have perfected for determining the nature and character of a diverse group of hazardous bacteria, viruses, and fungi. For bacterial and viral pathogens,...

Histological Technique

Animals were sacrificed by cervical dislocation. 2. Samples of the different organs from the tract were aseptically extracted (kidneys, urethers, bladders, and urethra), washed with saline solution, and immediately introduced into fixative. 3. Fixation was with 10 formaldehyde for at least 24 h (see Note 11). 4. The organ must be dehydrated before paraffin embedding, by transfer through increasingly graduated alcohols (see Notes 12 and 13), as follows d. Ethyl alcohol 96 , two passages of 1 h...

Testing Disinfectants in the Food Factory

Phenol Coefficient Method Anavella Gaitan Herrera Contamination of foods by the environment has direct public health and keeping quality significance. The food factory environment (with raw materials and processing) governs the numbers and types of microorganisms in finished products (1). Use of the appropriate sampling procedures permits us to discover the magnitude and type of contamination. Microbiological sampling allows objective evaluation of the disinfectants and the sanitation practices...

Microflora of the Gastrointestinal Tract

The mucosal surface of the human gastrointestinal (GI) tract is about 200-300 m2 and is colonized by 1013-14 bacteria of 400 different species and subspecies. Savage (1) has defined and categorized the gastrointestinal microflora into two types, autochthonous flora (indigenous flora) and allochthonous flora (transient flora). Autochthonous microorganisms colonize particular habitats, i.e., physical spaces in the GI tract, whereas allochthonous microorganisms cannot colonize particular habitats...

Adhesion Ability of Lactobacillus to Vaginal Epithelial Cells

Study by Microbiological Methods Virginia S. Oca a and Mar a Elena Nader-Mac as Adhesion of lactobacilli to the epithelium has been described as the first step in the formation of a barrier to prevent undesirable microbial colonization (1) consequently, it has been defined as a characteristic of interest for selecting probiotic strains (2-4). Several methods have been described to predict the adhesion ability of Lactobacillus. Early studies were phenomenological it was useful to determine...

Genotyping of Cryptosporidium parvum With Microsatellite Markers

Giovanni Widmer, Xiaochuan Feng, and Sultan Tanriverdi Recent outbreaks of cryptosporidiosis caused by Cryptosporidium parvum in the United States and other countries (1,2), as well as the emergence of cryptosporidiosis as a frequent cause of morbidity and mortality in immunodeficient individuals (3), have raised the interest of the research community in this parasite. The genus Cryptosporidium, phylum Apicomplexa, comprises an undefined number of species, of which only C. parvum is of public...

RNA Isolation Procedures for Rotavirus

Inactivation of Endogenous Ribonucleases 1. Prepare ribonuclease-free water by adjusting the volume of water required to contain 0.2 (w v) DEPC. 2. Incubate the treated water at 37 C for 12 h and then autoclave at 121 C for 30 min. 3. Immerse glassware and nonsterile plasticware in a solution of 0.2 (v v) DEPC and then autoclave at 121 C for 30 min. 4. Prepare buffers used in RNA procedures using DEPC-treated water. Buffers that cannot be autoclaved should be sterilized by filtration...

Effect of Lactobacilli Administration in the Vaginal Tract of Mice

Evaluation of Side Effects and Local Immune Response by Local Administration of Selected Strains Elisa Vinti i, Virginia Oca a and Mar a Elena Nader-Mac as Lactobacilli are the predominant microorganisms in the vaginal tract of human and some homeothermic animals (1). They can maintain the ecological equilibrium of the tract by protecting against pathogenic microorganisms (2). In the last few years, there has been an increased tendency to use probiotic microorganisms to restore the ecological...

Triglyceride Assay

Agar-Well Assay Technique 1. 0.2 M Phosphate buffer, pH 7.0, prepared as described in Subheading 2.2.1. 2. Substrate solution dissolve 0.25 mL triglyceride, 25 mg sodium azide, and 100 mL 0.2 M phosphate buffer, pH 7.0. 3. 1.5 Agar-Agar ultra pure (Merck). 1. 10 (w v) Gum arabic dissolve 10 g gum arabic in 100 mL dH2O. 2. 10 (v v) Triglycerides (substrate solution) Dissolve 10 mL triglycerides in 10 mL gum arabic. 3. 10 mM CaCl2 1.0 M Tris-HCl buffer, pH 7.0 dissolve 12.11 g Tris-HCl and...

Establish Documentation and Record Keeping Principle

Documentation and Record Keeping A record shows the process history, the monitoring, the deviations, and the corrective actions (including disposition of product) that occurred at the identified CCP. It may be in any form, e.g., processing chart, written record, computerized record. The importance of records to the HACCP system cannot be overemphasized. It is imperative that the producer maintain complete, current, properly filed, and accurate records. Four types of records should be...

Antimicrobial Assays

Microbial Culture Conditions The different strains must be cultured twice for activation in Erlenmeyer flasks (125 mL) containing 25 mL of Mueller-Hinton broth at 37 C for 12 h in a shaker, except for Lactobacillus rhamnosus, L. plantarum, Zymomonas mobilis, and Sac-charomyces cerevisiae (see Note 10). Stop the cultures at 0.8-1 OD600 (equivalent to 108 CFU ), dilute until 0.1 OD600, and use for antimicrobial testing (see Note 11). 3.3.2. Detection of Antimicrobial Activity in Solid...

Establish Verification Procedures Principle

The Codex guidelines define verification as the application of methods, procedures, tests and other evaluations, in addition to monitoring to determine compliance with the HACCP plan. Verification and auditing methods, procedures, and tests, including random sampling and analysis, can be used to determine whether the HACCP system is working correctly (20,47). 3.8.2. Description of Verification Activities Each HACCP plan should include verification procedures for individual CCPs and for the...

Determination of Bacterial Adhesion to Intestinal Mucus

Carlos Gusils, Vilma Morata, and Silvia Gonz lez The epithelial cells in the small intestine are covered by a relatively thick layer of mucus, secreted by specialized cells, which consists of mucin, many small associated proteins, glycoproteins, lipids, and glycolipids. The mucus contains receptors that recognize specific adhesion proteins (1,2). Adhesion or close association of bacteria to the epithelial cells may further contribute to competitive exclusion. In addition, bacterial adhesion to...

Microtechnique for Identification of Lactic Acid Bacteria

Carlos Gusils, Adriana P rez Chaia, Guillermo Oliver, and Silvia Gonz lez Certain gut species are pathogens, but a number of other resident bacteria may be of some benefit to host health. Examples include enterococci, lactobacilli, propionibacteria, and bifidobacteria, which are present in the colon in significant numbers. Identification and classification are not identical. A group can be identified only after it has been classified, based on a pattern of properties shown by all the members of...

Rflp

Prepare master mixture using the formula shown in Table 1, which is for one restriction digestion reaction (see Note 4). Transfer 30 L of master mixture to each tube, add 10 L of secondary PCR reaction to the tube, and mix well. Incubate in a 37 C waterbath for 2 h or overnight. Run electrophoresis on a 2 argrose gel with the entire 40 L from the restriction digestion reaction, using procedures standardized in the laboratory. Identify Cryptosporidium species and genotypes based on RFLP banding...

Growth Experiments

Inoculum Buildup for the Growth Experiments 1. Probiotic microorganisms Lactobacillus salivarius subsp. salivarius CRL 1328, L. crispatus CRL 1266, and L. paracasei subsp. paracasei CRL 1289. 2. LAPTg broth 15 g L peptone, 10 g L tryptone, 10 g L glucose, 10 g L yeast extract, and 1 mL L Tween-80, pH 6.5. Autoclave at 121 C for 15 min. Store at refrigeration temperature. The chemicals for LAPTg preparation were obtained from Britania (Argentina). 3. MRS broth Dehydrated MRS was obtained...

Dansyl Method see Note

Standards were derivatized prior to column injection as follows dansyl derivatization was performed with the addition of 100 L 2 N sodium hydroxide solution, 150 L saturated sodium bicarbonate and dansyl chloride solution. 2. The reaction mixture was incubated at 40 C for 45 min. 3. After incubation the residual dansyl chloride was removed by addition of 50 L ammonia. 4. After 20 min the sample was adjusted to 2.5 mL with acetonitrile. 5. The sample was filtered with a 0.22- m filter. 6. The...

T Nt T T T T T Nt

PCR amplification of the N-methyl transferase region of microcystin synthetase from an environmental bloom (22). Samples that proved toxic (T) or nontoxic (NT) by the phosphatase inhibition assay are indicated. (mcy) gene cluster of the strain PCC 7806 has been sequenced. It comprises 10 genes, encoding peptide synthetases, polyketide synthases, and tailoring functions. Two large bidirectionally transcribed operons (A-C and D-J) span a region of 55 kb. Further knockout mutations have...

PCR Methods

Reverse Transcriptase (RT)-Mediated PCR 1. Denature 1-5 L of the dsRNA template (see Note 8) by heating to 97 C in the presence of 3.5 L DMSO for 5 min (see Note 9). Place the reaction tubes immediately on ice. 2. To amplify full-length gene segment 9 (1062 bp) encoding VP7, assemble a PCR reaction cocktail containing the following 5 L 10X PCR buffer, 8 L of the working dNTP mix (final concentration of each dATP, dCTP, dGTP, dTTP was 200 M), 1.5 mM MgCl2, 10 pmol of each primer (Table...

I

An agarose gel (1.5 w v) showing the DNA profiles of a collection of unrelated Campylobacter spp. isolates. Fig. 3. MAMA-PCR showing a 192-bp DNA fragment corresponding to the wild-type DNA gyrase in C. coli (A) and a 265-bp fragment corresponding to the mutated gene in C. jejuni (B). Fig. 3. MAMA-PCR showing a 192-bp DNA fragment corresponding to the wild-type DNA gyrase in C. coli (A) and a 265-bp fragment corresponding to the mutated gene in C. jejuni (B). Fig. 4. Amplified gene...

Putting Water Throughtap In Vagina

H, inoculation of 0.5 mg of estradiol valeratel C, control mice inoculated with peptone water without lactobacilli L, inoculation of lactobacilli in peptone water S, days of mice killing and sample processing. H, inoculation of 0.5 mg of estradiol valeratel C, control mice inoculated with peptone water without lactobacilli L, inoculation of lactobacilli in peptone water S, days of mice killing and sample processing. 9. Immunofluorescence assays were carried out to evaluate whether local...

Extraction of DNA

Extraction of Fecal and Environmental DNA DNA extracted from feces or water pellets containing Cryptosporidium oocysts is often suitable for PCR detection and genotyping. Since PCR inhibitors may copurify with environmental DNA (16,17), some investigators prefer to purify the oocysts first and then extract DNA from the oocysts. Using purified or partially purified oocysts may also be an advantage when working with a sample containing low concentrations of oocysts. Methods for purifying...

Differentiation of Lactic Acid Bacteria Strains by Postelectrophoretic Detection of Esterases

Roxana Beatriz Medina, Marta Beatriz Katz, and Silvia Gonz lez Lactic acid bacteria (LAB) comprise a diverse group of Gram-positive, non-spore-forming microorganisms. These bacteria are widely used in food technology. The species identification of LAB depends mainly on physiological and biochemical criteria. The esterolytic systems of LAB remain poorly characterized. Esterases (EC 3.1.1.3) represent a diverse group of hydrolases catalyzing the cleavage and formation of esters bonds (1)...

Integrated Cell CulturePCR for Detection of Enteric Viruses in Environmental Samples

Recently, an integrated cell culture polymerase chain reaction (ICC PCR) technique has been developed for the detection of viruses in environmental samples providing a reliable method for practical analysis and direct monitoring of environmental samples for viral pathogens (1,2). CC PCR allows for detection of infectious viruses in hours to days compared with the days or weeks necessary with cell culture alone. Bacterial indicator organisms are commonly used to evaluate environmental samples...

Microbiological Analysis of Cosmetics Anavella Gaitan Herrera

Cosmetics are products of chemical or natural origin dedicated specifically for use in skin and mucosa (1). The constant development of the cosmetic industry has generated the necessity to carry out microbiological analysis on the raw materials used in the industrial production of cosmetics as well as the final products, with the purpose of obtaining products of good microbiological quality (2). Cosmetic products are recognized to be substrates for the survival and development of a large...

Microcystis Nodularia and Cylindrospermopsis

Microcystin Structure

Saker, Michelle C. Moffitt, and Brett A. Neilan Cyanobacteria are ubiquitous in the freshwater environment. Their success as a group in a wide range of aquatic habitats has been attributed to their unique physiological characteristics and their high adaptive ability over a wide range of environmental conditions. They are capable of reaching very high biomass levels, often dominating the other aquatic biota, and under some circumstances can accumulate near the water...

Public Health Implications Related to Spread of Pathogens in Manure From Livestock and Poultry Operations

During the 20th century, food animal agriculture grew from small operations, where livestock (cattle, sheep, and swine) and poultry (chickens and turkeys) had access to free range, to large operations where animals and poultry were concentrated and confined to feed lots or buildings. The quantity of manure produced by confinement animals in the United States has been estimated to be in excess of 61 million tons of dry matter per year (1), and another report states that 1.2 billion tons of...

Meat Model System Development for Antibacterial Activity Determination

Graciela Vignolo and Patricia Castellano Bacteriocins are antibacterial substances produced by many different bacterial species. Although the bacteriocins form a heterogenous group with respect to production of bacteria, antibacterial spectrum, mode of action, and chemical properties, they are by definition proteinaceous compounds that are bactericidal toward organisms taxo-nomically close to the producer (1). The ability of lactic acid bacteria (LAB) to compete and finally dominate in mixed...

The Hazard Analysis and Critical Control Point System in Food Safety

The Hazard Analysis and Critical Control Point (HACCP) system is a preventive method of ensuring food safety. Its objectives are the identification of consumer safety hazards that can occur in the production line and the establishment of a control process to guarantee a safer product for the consumer it is based on the identification of potential hazards to food safety and on measures aimed at preventing these hazards. HACCP is the system of choice in the management of food safety (1-3). The...

Purification of Bacteriocins Produced by Lactic Acid Bacteria

Lucila Saavedra, Patricia Castellano, and Fernando Sesma Bacteriocins are antibacterial substances of a proteinaceous nature that are produced by different bacterial species. Lactic acid bacteria (LAB) produce biologically active peptides or protein complexes that display a bactericidal mode of action almost exclusively toward Gram-positive bacteria and particularly toward closely related species. Generally they are active against food spoilage and foodborne pathogenic microorganisms including...

Determination of Aflatoxins and Zearalenone in Different Culture Media

Dante Javier Bueno and Guillermo Oliver Some molds produce desirable changes in food, but most are merely esthetically undesirable. There has also been an increasing awareness that certain metabolic products of some molds commonly found on foods and feed are dangerous to humans and animals. These toxin substances, mycotoxins, are secondary metabolites produced by different fungi, especially Aspergillus, Penicillium, Fusarium, and, to a lesser degree, Alternaria (1). The most important toxins...

Screening of Hydrogen Peroxide Generating Lactic Acid Bacteria

Qualitative Method on Plate 1. Solution A was prepared with 12.5 mg of TMB (see Note 3) and 3 mL of methanol (see Note 4 17.34 mM TMB), with shaking to complete dissolution. 2. Solution B was prepared with 0.5 mg of peroxidase and 1 mL sterilized distilled water (0.5 mg mL or 100 U mL peroxidase see Note 5). 3. Preparation of TMB-agar plates MRS medium was melted at 45 C. Then 10 mL of MRS agar were mixed with a 0.6-mL aliquot of solution A and a 0.2-mL aliquot of solution B. This...

Assessing Survival of Dairy Propionibacteria in Gastrointestinal Conditions and Adherence to Intestinal Epithelia

Gabriela Z rate, Silvia Gonz lez, and Adriana P rez Chaia The genus Propionibacterium consists of two principal groups, cutaneous and classical or dairy. Cutaneous species are predominant members of the microbial population of human skin and have also been isolated from the feces of humans and other vertebrate animals. They are often considered opportunistic organisms and have been occasionally associated with infections in humans (1). Dairy propionibacteria are microorganisms extensively used...

Nontraditional Method of Evaluating Disinfectants

Evaluation Disinfectant

With Isolated Microorganisms From the Food Factory Anavella Gaitan Herrera Cleaning and disinfection in the food industry are critical in the production process, and the efficacy of the disinfectants used is frequently debated (1). Several factors are involved in the effectiveness of a disinfectant agent. It is important to consider the number and type of microorganism present as well as the physical and chemical characteristics of the water these factors vary from industry to industry and they...

Immunomagnetic Separation of Pathogenic Organisms From Environmental Matrices

Stadterman-Knauer One of the most difficult challenges in the analysis of environmental samples is to separate the organism of interest from a sample that is high in background debris. Immunomagnetic separation (IMS) is one technique that has been developed to accomplish this in a rapid and reliable assay. Immunomagnetic separation (or biomagnetic separation) involves a superparamag-netic, monodispersed, polystyrene microsphere that is coated with a specific...

Bacteriocin Purification

Obtaining the Crude Extract 2.2.2. Solid-Phase Extraction Protocols 1. Octadecyl (C18) Impaq RG1080 columns, pore diameter 100 A, particle size 80 m (Sigma), or C8 columns from Supelco (Supelclean LC-8) with 3-mL tubes (or similar see Note 2). 2. Acetonitrile and isopropanol (HPLC grade Merck). 3. Turvo-Vap Evaporator (Zymark, Hopkinton, MA). 2.2.3. Ion Exchange Chromatography 1. SP-Sepharose Fast Flow strong cation exchanger (Amersham Pharmacia Biotech). 2. 0.1 M Phosphate buffer, pH...

Molecular Genotyping of Irish Rotavirus Strains Fiona OHalloran and Seamus Fanning

Rotavirus is the primary etiological agent of gastroenteritis in infants and young children worldwide (1). In developing countries, it is estimated that rotavirus is responsible for one-third of all diarrhea-associated hospitalizations and 873,000 deaths annually (2,3). In industrialized countries, where mortality from rotavirus is low, infection is widespread, and nearly all children experience an episode of rotavirus diarrhea in the first 3-5 yr of life (4,5). Rotaviruses have important...

Characterization of the Bacteriocin

Among 134 vaginal lactobacilli, Lactobacillus salivarius subsp. salivarius CRL 1328 (CERELA Culture Collection) was selected for further study because it was able to inhibit the growth of Enterococcus, Listeria, Neisseria, and Gardnerella strains in the plaque assay for a substance different from acid or hydrogen peroxide. 1. Heat resistance of the antimicrobial substance is studied by heating aliquots of the filter-sterilized supernatant to 60 C, 80 C, and 100 C for 10 min and also after...

Molecular Aspects of Disease Pathogenesis in the Transmissible Spongiform Encephalopathies

The transmissible spongiform encephalopathy (TSE) diseases are a group of rare, fatal, and transmissible neurodegenerative diseases that include kuru and CreutzfeldtJakob disease (CJD) in humans, scrapie in sheep, transmissible mink encephalopathy (TME 82), and chronic wasting disease (CWD) in mule deer (141) and elk (142). Over the last 20 yr, they have gone from a fascinating but relatively obscure group of diseases to one that is a major agricultural and economic problem as well as a threat...

Establish a Monitoring System for Each Critical Control Point Principle

The Codex Hazard Analysis and Critical Control Point (HACCP) System and Guidelines for Its Application defines monitoring as the act of conducting a planned sequence of observations or measurements of control parameters to assess whether a CCP is under control. Monitoring procedures performed during the operation should result in written documentation that will serve as an accurate record of the operating conditions. 3.6.2. Design of a Monitoring System The control measures are intended to...

Colonization Capability of Lactobacilli and Pathogens in the Respiratory Tract of Mice

Microbiological, Cytological, Structural, and Ultrastructural Studies Rosa Cangemi de Gutierrez, Viviana M. Santos, and Mar a Elena Nader-Mac as Respiratory tract infections are among the bacterial infections that affect humans with higher frequency. Those produced by Streptococcus pneumoniae are reported to have the highest incidence in the world, affecting both children and old people. As a 2001 report from the World Health Organization I expressed it, the basic fight of children under 5 yr...

John F T Spencer Alicia L Ragout de Spencer

Planta Piloto de Procesos Industriales Microbiol gicos PROIMI -CONICET San Miguel de Tucum n, Argentina 2004 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology is a trademark of The Humana...

PrPres and TSE Species Barriers

Species barriers in the TSE are defined as the resistance to TSE disease by one species following infection with the TSE agent of another species see ref. 93 for review . This resistance is reflected by extremely long incubation times upon first passage of a TSE agent into a new host species. The barrier to infection upon first exposure can be very strong and can lead to incubation times that exceed the lifetime of the host. However, as the agent is serially passaged through the new host,...

Spectrophotometric Determination of Histamine in Fisheries Using an Enzyme Immunoassay Method

Fontana, and Marcos L. Pessatti The biogenic amines are low-weight organic bases that exhibit variable biological activity. Approximately 30 vasoactive and psycoactive amines have been found in foodstuffs 1 . Histamine, tyramine, phenylethylamine, tryptamine, cadaverine, putrescine, spermine, and spermidine are examples of biogenic amines detectable in meat and its derivatives 2 . These amines occur naturally in animals, plants, and microorganisms owing to their...

Animal Model for In Vivo Evaluation of Cholesterol Reduction by Lactic Acid Bacteria

Mar a P a Taranto, Gabriela Perdig n, Marta M dici, and Graciela Font de Valdez For many years, it has been recognized that elevated serum cholesterol is a risk factor associated with atherosclerosis and coronary heart disease, the latter being a major cause of death in Western countries. Numerous drugs that lower cholesterol have been used to treat hypocholesterolemic individuals 1 . However, the undesirable side effects of these compounds have caused concerns about their therapeutic use 2 ....

Detection of Tetracycline Resistance Genes by PCR Methods

Chee-Sanford, Natalie Garrigues, Asma Mehboob, and Roderick I. Mackie Rapid, accurate, and sensitive determination of antibiotic resistance profiles of various human and animal pathogens becomes a vital prerequisite for successful therapeutic intervention in the face of the increased occurrences of drug-resistant bacterial infections. The current methods, which are dependent on cultivation of pathogens and phenotypic expression of antibiotic resistance, usually...

Mono Mac 6 Cells and Acanthamoeba castellanii as Suitable In Vitro Models

Intracellular Multiplication of Legionellae Within Human Monocytes Legionellae are important etiological agents of pneumonia. Legionella pneumophila predominantly serogroup 1 is detected in most cases of legionellosis other species only occasionally cause infections, predominantly in immunocompromized patients 1-4 . Aquiferous technical systems are the primary source of infection air-conditioning systems, refrigerators, showers, whirlpools, springs, taps, moisturizing equipment, medical...

Kinetics of Hydrogen Peroxide Production by Lactobacilli

Inoculum Buildup for the Growth Experiments 1. Lactobacilli subcultivation L. crispatus CRL 1266, L. paracasei CRL 1251 and 1289 from human vagina , and L. acidophilus 1012 and 2014 from bovine vagina . These Lactobacillus strains were selected for hydrogen peroxide production and inhibition of pathogenic microorganisms, according to the screening performed. The lactobacilli were grown as described in Subheading 3.1.2., step 1. 2. The third culture was centrifuged 10 min, 2000g , and the...

Chromogenic Method

Substrates a- and -Naphthyl Derivatives of Fatty Acids 1. 0.1 M Sodium phosphate buffer, pH 7.0. a. Solution A 0.2 M NaH2PO4 Weigh 27.6 g NaH2PO4-H2O and make to 100 mL with distilled water dH2O . b. Solution B 0.2 MNa2HPO4 Weigh 53.05 g of Na2HPO4-7H2O and make to 100 mL with dH2O. c. To prepare 1 L of 0.1 M phosphate buffer pH 7.0 , mix 195 mL of solution A and 305 mL of solution B and bring to 1000 mL with dH2O. 2. 66 mM a- and -naphthyl substrate in ethanol water 1 1 . Always use a...

Direct Microscopic Somatic Cell Count

Slide Identification and Sample Preparation 1. Identify each sample area legibly and indelibly on the margin of slide. 2. Mix samples with a vortex 25 times. 3. Heat samples in a water bath to 30-40 C. 5. Mix them carefully and cool to the temperature at which the microsyringe has been calibrated. 3.2.2. Sample Measurement and Smear Preparation 1. Place 0.01 mL of milk sample on a clean slide in a 1-cm2 area. 2. Holding the microsyringe vertical, place the tip near the center of the area...

Flavonoids From Argentine Tagetes Asteraceae With Antimicrobial Activity

Flavonoid Antimicrobial Effect Image

Baigon, Lucia I. C. de Figueroa, and Lidia R. Abdala The flavonoids, constituting one of the most numerous and widespread groups of natural plant constituents, are important to humans not only because they contribute to plant colors but also because many members are physiologically active. These low-molecular-weight substances, found in all vascular plants, are phenylbenzopyrones. Over 4000 structures have been identified in plant sources, and they are categorized...

Differences Between Biogenic Amine Detection by HPLC Methods Using OPA and Dansyl Derivates

Arena, and Mar a C. Manca de Nadra Biogenic amines can be formed and degraded as a result of normal metabolic activity in animals, plants, and microorganisms and are usually produced by the decarboxylation of amino acids 1 . Recent trends in food security are promoting an increasing search for trace compounds that can affect human health. Although they are present in fermented foods and beverages in low quantities, they exhibit interactions with normal human...

Molecular Based Identification and Typing of Campylobacter jejuni and C coli

Brigid Lucey, Fiona O'Halloran, and Seamus Fanning Thermophilic Campylobacter spp., mainly Campylobacter jejuni and to a lesser extent C. coli are recognized as the most common bacteriological causes of gastroenteritis in humans 1 . As enteric infection with Campylobacter organisms cannot be distinguished from that caused by other enteric pathogens, a definitive diagnosis can only be made by isolating or detecting the organism from the feces. The epidemiology of Campylobacter enteritis has been...

Salt Aggregation Test

Bacterial cell suspension preparation. The cultures were centrifuged at 2000g for 10 min, and the cells were washed with saline and resuspended in phosphate buffer 0.02 M, pH 6.8 to a final concentration of 1 x 109 CFU mL see Note 2 . 2. Salt aggregation assay. The aggregating properties of lactobacilli in NH4 2SO4 were tested by mixing on a glass slide 25 L of the bacterial suspension and the same volume of each one of the salt solutions. They were mixed with a circular movement for 2 min,...

Integron Analysis and Genetic Mapping of Antimicrobial Resistance Genes in Salmonella enterica serotype Typhimurium

Antimicrobial resistance determinants may be transferred among bacteria via mobile genetic elements including plasmids, transposons, and the more recently explored integrons 1 . Integrons are naturally occurring genetic elements found as part of the Tn21 transposon family or located on various broad host-range plasmids 2 . The fundamental integron structure consists of a 5'-conserved segment 5'-CS of 1.4-kbp and a 2-kbp 3'-CS 3 . Between these conserved regions are DNA sequences of variable...

Estimation of Growth Parameters

Description of the Bacterial Growth Curves A modified four-parameter-Gompertz model was used 10 . 2.3.2. Method for Estimation of the Growth Curves 1. Ordinary least squares Statistical software STATISTICA 5.0. STATISTICA for Windows Computer program manual , Stat Soft Inc., 1995 . For the algorithm of minimization of the loss function, the Rosenbrock quasi-Newton procedure was applied. 2. Bootstrap method SPSS 10 for Windows and S-PLUS 200. To plot the growth curves and also to estimate...

RNA Extraction Procedure Based on the Boom Method 15 see Note

Add 40 L of silica solution to 900 L of L6 buffer in a 1.5-mL microcentrifuge tube see Notes 10 and 11 . 3. Vortex the mixture for 5 s, keep it at room temperature for 10 min, and mix again for 5 s. 4. Centrifuge at 12,000g for 15 s and discard the supernatant see Notes 10 and 13 . 5. Wash the silica pellet twice with 900 L of L2 buffer, twice with 900 L of 70 ethanol, and once with 900 L of acetone. Centrifuge for 15 s at 12,000g after each wash and discard the supernatants see Notes 10,...

Contributors

Francesc-Xavier Abad Enteric Virus Laboratory, Department of Microbiology, School of Biology, University of Barcelona, Barcelona, Spain Lidia R. Abdala Facultad de Ciencias Naturales e Instistuto Miguel Lillo, Universidad Nacional de Tucum n, Tucum n, Argentina Donald G. Ahearn Environmental Research Center, Department of Biology, Georgia State University, Atlanta, GA Mar a R. Alberto Centro de Referencia para Lactobacilos CERELA - CONICET, Tucum n, Argentina Juan Alvarez Departamento de...

Microbiological Test for Sanitation of Equipment in the Food Factory

Microbiological sampling of utensils, tableware, and kitchenware, in addition to equipment, permits objective evaluation of sanitation practices and procedures used for these items from food service operations 1 . 3. Stock phosphate buffer solution. 5. Plate count agar PCA , Violet Red Bile agar VRBA , MF Endo broth. 6. Buffered rinse solution add 1.25 mL of stock phosphate buffer solution, 5 mL of 10 aqueous sodium thiosulfate Na2S2O3-5H2O , 4 g azolectin see Note 1 , and 10 g Tween-20 or...

Sampling Procedure 3458

Open the sterile swab see Note 3 , grasp the end of a stick, not touching any portion that might be inserted into the vial, and remove the swab aseptically see Note 4 . 2. Open a buffered rinse solution, and moisten the swab head pressing out the excess solution against the interior wall of the vial with a rotating motion . 3. Make a 30 angle contact with the surface. Rub the swab head slowly and thoroughly over 50 cm2 of surface three times, reversing direction between successive strokes....

Separation of Flavonoids

Separation of Flavonoids by Paper Chromatography 34-38 1. Dissolve about 1 g of the dried residue in 80 aqueous methanol 1 mL and spot it on the lower right-hand corner of a sheet of Whatman 3 MM chromatographic paper semipreparative . Use a flow of warm air for solvent evaporation between repeated applications of the solutions to the paper. The final spot, which appears deep purple when viewed under a 366-nm UV lamp, is about 2 cm in diameter and 8 cm from each edge of the paper. 2....

Bacterial Surface Characteristics Applied to Selection of Probiotic Microorganisms

Oca a, and Mar a Elena Nader-Mac as For the study of probiotic microorganisms, the in vitro selection tests need to be based on a solid scientific foundation 1 . Surface characteristics, one of the in vitro properties are used to evaluate the potentially probiotic strains of lactobacilli 2 . Bacterial surface properties have been associated with attachment to a variety of substrata. Bacterial adhesion to tissues is considered the first step, and such adhesion...

Purification of Antilisterial Bacteriocins Jean Marc Berjeaud and Yves Cenatiempo

In recent years, numerous contamination outbreaks, involving various pathogens i.e., Listeria and Salmonella , have increased concern over food preservation. Research efforts have focused on the discovery of new molecules targeting such foodborne pathogens and therefore able to inhibit and or kill them. Lactic acid bacteria LAB extensively used in fermented foods for thousands of years not only improve their flavor and texture but also inhibit pathogenic and spoilage microorganisms. LAB...